1. Field of the Invention
This invention relates to novel methods and pharmaceutical formulations for treating atheroma, tumors and other neoplastic tissue, as well as other conditions that are responsive to the induction of targeted oxidative stress. This invention also relates to novel methods for determining the radiation sensitization potential of a compound.
2. Publications Cited by Reference
Certain publications are cited in this application through the use of the following superscript numbers:
1 Buettner, et al., Radiation Research, Catalytic Metals, Ascorbate and Free Radicals: Combinations to Avoid, 145:532-541 (1996)
2 Isoda, et al., J. Cancer Research, Change in Ascorbate Radical Production in an Irradiated Experimental Tumor with Increased Tumor Size, 56:5741-5744 (1996)
3 Riley, Int. J. Radiat. Biol., Free Radical in biology: oxidative stress and the effects of ionizing radiation, 65(1):27-33 (1994)
4 Sessler, et al., J. Phys. Chem. A, One-Electron Reduction and Oxidation Studies of the Radiations Sensitizer Gadolinium (III) Texaphyrin (PCI-120) and Other Water Soluble Metallotexaphyrins, 103: 787-794 (1999)
5 Adams, et al., Radiation Res., 67:9-20 (1976)
6 Riley, Int. J. Radiat. Biol., Free Radicals in Biology: Oxidative Stress and the Effects of Ionizing Radiation, 65(1):27-33 (1994)
7 Magda, et al., U.S. Pat. No. 5,798,491, Multi-Mechanistic Chemical Cleavage Using Certain Metal Complexes, issued Aug. 25, 1999
8 Young, et al., U.S. Pat. No. 5,776,925, Methods for Cancer Chemosensitization, issued Jul. 7, 1998
9 Sessler, et al., U.S. Pat. No. 5,622,946, Radiation Sensitization Using Texaphyrins, issued Apr. 22, 1997
10 Sessler, et al., U.S. Pat. No. 5,457,183, Hydroxylated Texaphyrins, issued Oct. 10, 1995
11 Sessler, et al., Accounts of Chem. Res., Texaphyrins: Synthesis and Applications, 27:43-50 (1994)
12 Hemmi, et al., U.S. Pat. No. 5,599,928, Texaphyrin Compounds Having Improved Functionalization, issued Feb. 4,1997
13 Young, et al., Investigative Radiology, 29:330-338 (1994)
14 Mosmann, J. Immunol. Methods, 65:55-63 (1983)
15 Lin, et al., Analytical Biochemistry, The Cytotoxic Activity of Hematoheme: Evidence for Two Different Mechanisms, 161:323-331 (1987)
16 Volpin, et al., WO097/03666, EP 0 786 253 A1, U.S. Pat. No. 6,004,953, Agent for Suppressing Tumor Growth
All of the above publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.
3. Background Information
The treatment of solid mammalian tumors with Ionizing radiation involves the in situ generation of hydroxyl radicals and other reactive oxygen species which, due to the focusability of the ionizing radiation are primarily located in the tumor, i.e., in tumor cells. These reactive species possess extreme oxidizing properties which oxidize biomolecules in vivo thereby interfering with cellular metabolism.1 For example, it is reported that ionizing radiation, such as X-rays and xcex3-rays, induces irreversible damage to cellular DNA through production of hydroxyl radicals and other reactive oxygen species in the cell leading to cell death2.3 or initiation of the mechanism of apoptosis.4 
One generally accepted mechanism of the cellular effect of ionizing radiation is initial damage inflicted to the cell""s DNA by reactive oxygen species generated by the ionizing radiation. In the presence of molecular oxygen, this damage is largely irreparable. Contrarily, in the absence of molecular oxygen (such as hypoxic cells), cellular antioxidants such as ascorbate and NAD(P)H can act to repair damage to the tumor DNA.
Tumor treatment via the use of ionizing radiation can be enhanced by increasing the radiosensitivity of the tumor cells. One method suggested for enhancing radiosensitivity has been the external administration of a compound having a high affinity for electrons, which ideally localizes in the tumor. Proposed radiation sensitizers include compounds such as halogenated pyrimidines, nitroimidazoles and gadolinium (III) complexes of the pentadentate macrocycle texaphyrin.4 Motexafon gadolinium (a gadolinium (III) texaphyrin complex) is currently in Phase III clinical trials for the treatment of brain metastases.4 
Phthalocyanine and naphthalocyanine polydentate ligands of the transition metals cobalt and iron have been described as suppressing the growth of tumor cells when administered in combination with a biogenic reductant such as ascorbic acid.16
The observation that radiation sensitization occurs as a function of redox potential gave rise to the proposal that such compounds function by interception of aqueous electrons, thus preventing their recombination with cytotoxic radicals.5 Subsequent evidence showing a lack of radiation sensitization activity for lutetium (III) texaphyrin in animal models notwithstanding the rapidity of reaction between this complex and hydroxyl radicals under pulsed radiolytic conditions and minimal apparent nuclear localization suggest that this proposal might not fully explain the mechanism by which the gadolinium texaphyrins act as radiosenstizers.4
In view of the above, an understanding of the mechanism for radiosentization of tumor cells would be particularly helpful. Such an understanding could be used for testing in the discovery of new compounds useful as radiation sensitizers as well as in maximizing the therapeutic effect achieved by use of such compounds in the presence or the absense of ionizing radiation.
This invention is premised upon the unexpected observation that the known motexafin gadolium radiation sensitizer acts to catalyze the oxidation of NAD(P)H, ascorbate and other reducing agents under approximate physiological conditions, leading to reactive oxygen species generation. Depletion of these reducing agents will inhibit biochemical pathways that in vivo utilize reducing agents to effect repair of the damage inflicted by reactive oxygen species. Additionally, since hydrogen peroxide is recognized as probably the most significant of the reactive oxygen species6, the generation of hydrogen peroxide will facilitate oxidative attack on the tumor or other tissue where it is produced.
Moreover, this discovery that motexafin gadolium acts to catalyze the oxidation of reducing agents under approximate physiological conditions to produce one or more reactive oxygen species such as superoxide and hydrogen peroxide, serves as a basis to assess the radiation sensitizing potential of other compounds. Specifically, a candidate compound can be screened to determine its ability to generate one or more reactive oxygen species under appropriate conditions. In turn, the amount of reactive oxygen species so produced is then correlated to the radiation sensitizing ability of the compound. Accordingly, in one of its method aspects, this invention is directed to a method for determining the radiation sensitization potential of a compound by the steps of:
a) introducing a compound to be tested into an aqueous solution of a cellular metabolite having a standard biochemical reduction potential more negative than the standard biochemical reduction of oxygen/hydrogen peroxide;
b) monitoring the solution for the occurrence of a reaction that produces one or more reactive oxygen species; and
c) determining whether the compound has potential radiation sensitization activity, wherein the potential for radiation sensitization activity correlates to the occurrence of a reaction that produces one or more reactive oxygen species.
The reaction that produces a reactive oxygen species can be monitored in numerous manners as is well known to the skilled artisan, by measuring one or more of: depletion of oxygen, production of hydrogen peroxide, decreased concentration of the cellular metabolite, or generation of an oxidation product of the cellular metabolite. The cellular metabolite is preferably a compound selected from the group consisting of ascorbate, NADPH, NADH, FADH2 and reduced glutathione. Even more preferably, the cellular metabolite is ascorbate or NADPH.
In another embodiment, a compound which is determined to have radiation sensitization potential by a method of the present invention can be administered to a mammalian host bearing a tumor and the tumor is subsequently exposed to ionizing radiation.
In still another embodiment there is provided a method for killing a tumor cells by:
a) administering to the tumor cell a compound (other than a texaphyrin) that catalyzes the production of one or more reactive oxygen species from a cellular metabolite having a standard biochemical reduction potential more negative than the standard biochemical reduction of oxygen/hydrogen peroxide; and
b) exposing the cell to ionizing radiation. Preferably, the compound preferentially accumulates in tumor cells, e.g., as do porphyrin derivatives. One preferred compound is Fe(III) porphyrin.
The knowledge that certain compounds exhibiting radiation sensitizing properties can catalytically effect the production of one or more reactive oxygen species from cellular metabolites having a biochemical reduction potential more negative than oxygen/hydrogen peroxide also serves as a basis for a method to enhance the rate of tumor cell death by co-administration of a thiol-depleting compound to the cell. Such a thiol-depleting compound will reduce the level of thiol reducing agents such as glutathione thereby removing essential components of the metabolic pathways for repairing the cellular damage generated by the reactive oxygen species.
Accordingly, in another of its method aspects, this invention is directed to a method for killing a tumor cell, which method comprises:
a) selecting a compound having radiation sensitization potential as per above;
b) administering said compound to the tumor cell; and
c) co-administering to the tumor cell a thiol-depleting agent. Preferably, the radiation sensitizing compound selected in step a) above is a texaphyrin and the thiol-depleting agent is buthionine sulfoximine.
The above method has applicability in the treatment of cancer in a patient. When so applied, this invention provides for a method of treatment of cancer comprising administering to a patient suffering therewith an effective amount of a texaphyrin radiation sensitzer, an effective amount of a thiol-depleting agent, and an effective amount of ionizing radiation.
In yet another of its method aspects, this invention is directed to a method for killing a tumor cell, which method comprises:
a) administering to said cell a compound that catalyzes the production of one or more reactive oxygen species from a cellular metabolite having a standard biochemical reduction potential more negative than the standard biochemical reduction of oxygen/hydrogen peroxide; and
b) co-administering to said cell a second agent selected from the group consisting of DNA alkylators, topoisomerase inhibitors, redox cycling agents, thiol-depleting agents, metabolic inhibitors, and mitochondrial inhibitors. Preferred DNA alkylators include carmustine. Preferred redox cycling agents include alloxan, phenazine methosulfate, menadione, copper/putrescine/pyridine, methylene blue, paraquat, doxorubicin, bleomycin, and ruthenium (II) tris-(1 ,10-phenanthroline-5,6-dione). Preferrred thiol-depleting agents include buthionine sulfoximine and diethyl maleate. Preferred metabolic inhibitors include folic acid analogs (e.g., methotrexate and trimetrexate), pyrimidine analogs (e.g., fluorouracil, floxuridine, cytarabine and azacitidine), and purine analogs and related inhibitors (e.g., mercaptopurine, thioguanine, pentostatin and fludarabine). Preferred mitochondrial inhibitors include oligomycin and antimycin A. In another preferred embodiment, this method for killing tumor cells includes the additional step of exposing the cell to ionizing radiation.
In another, but related aspect of the invention, a first agent selected from the group consisting of DNA alkylators, topoisomerase inhibitors, redox cycling agents, thiol-depleting agents, metabolic inhibitors, and mitochondrial inhibitors is co-administered with a second agent that catalyzes the production of one or more reactive oxygen species from a cellular metabolite having a standard biochemical reduction potential more negative than the standard biochemical reduction of oxygen/hydrogen peroxide, to a subject having a condition (other than a tumor or atheroma) typically treated with such first agent.
Still further, in another aspect of this invention, there is provided a method of selectively killing cells in a mammalian host bearing a tumor or atheroma, which method comprises:
a) administering to said mammalian host an agent that catalyzes the production of one or more reactive oxygen species from an intracellular reducing agent, preferably ascorbate or NAD(P)H;
b) optionally, allowing sufficient time for said agent to preferentially accumulate in the cells of the tumor or atheroma; and c) administering to said mammalian host a source or precursor of the reducing agent such as to increase the reactive oxygen species production in the tumor or atheroma.
In one preferred embodiment, this method further includes the steps of exposing the tumor or atheroma to ionizing radiation. In another preferred embodiment, the agent employed in this method is motexafin gadolium or motexafin lutetium or combinations thereof.
In yet another aspect of this invention, there is provided a method of treating a mammalian host bearing a tumor or an atheroma comprising administering to that host a therapeutically effective amount of a combination of motexafin gadolinium and motexafin lutetium and exposing the tumor or atheroma to ionizing radiation.
In one of its composition aspects, this invention is directed to pharmaceutical compositions for selectively killing cells in a host bearing a tumor or atheroma comprising a pharmaceutically acceptable carrier and an effective amount of an agent that catalyzes the production of one or more reactive oxygen species from an intracellular reducing agent provided that said agent is not a texaphyrin.
In another of its composition aspects, the invention encompasses an agent that preferentially accumulates in tumor or atheroma cells and catalyzes the production of one or more reactive oxygen species from a cellular metabolite having a standard biochemical reduction potential more negative than the standard biochemical reduction of oxygen/hydrogen peroxide, a source or precursor of the cellular metabolite, and a pharmaceutically acceptable excipient.
Still further, this invention provides for use of an agent that catalyzes the production of one or more reactive oxygen species from an intracellular reducing agent in the treatment of mammalian tumors or atheroma.